Diversity of bacteriophages in the ancient human microbiome

Project Information

ARCHAIC

Grant agreement ID: 101111040

DOI

10.3030/101111040

Project closed

EC signature date 7 April 2023

Start date 1 May 2023

End date 30 April 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 199 694,40

Coordinated by

UNIVERSITY COLLEGE CORK - NATIONAL UNIVERSITY OF IRELAND, CORK
Ireland

Periodic Reporting for period 1 - ARCHAIC (Diversity of bacteriophages in the ancient human microbiome)

Reporting period: 2023-05-01 to 2025-04-30

Summary of the context and overall objectives of the project

The ARCHAIC project addressed a critical gap in microbiome research: the lack of evolutionary insight into the human virome, specifically bacteriophages (phages), through deep-time metagenomic analyses. Previous paleogenomic efforts primarily focused on bacterial components, leaving viral fractions, especially phages, largely unexplored. Given the central role of phages in shaping microbial communities and influencing host-microbe interactions, ARCHAIC set out to reconstruct phage diversity, persistence, and evolution using ancient gut metagenomes.

Using state-of-the-art bioinformatic and molecular methods, the project focused on high-quality ancient DNA extracted from paleofeces across multiple archaeological sites and historical periods. The primary objective was to trace the diversity and dynamics of viral communities over time, with an emphasis on highly abundant and conserved phage groups such as crAssphages.

A key innovation of the project was the benchmarking and optimization of taxonomic classifiers tailored for ancient viral metagenomes, ensuring robust detection even in highly degraded samples. By integrating ecological, evolutionary, and computational approaches, ARCHAIC contributes to broader discussions on microbial co-evolution, ancestral microbiota baselines, and the long-term dynamics of phage-bacterial relationships in human-associated ecosystems.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Over the course of the project, we processed and analyzed 20 publicly ancient gut metagenomes, using tailored pipelines to retrieve and validate phage sequences. The analytical workflow involved raw read quality control, de novo assembly (MEGAHIT, metaSPAdes), taxonomic classification (Centrifuge, Kraken2, KrakenUniq, Metabuli), and manual validation through DNA damage profiling and reference genome matching.

A significant innovation was the benchmarking of classification tools under ancient DNA conditions, revealing the limitations of existing profilers and the importance of using targeted viral databases. We also tested multiple binning tools (VAMB, SemiBin2, taxVAMB) to reconstruct viral metagenome-assembled genomes (vMAGs), achieving unprecedented resolution in ancient virome reconstruction.

Key achievements include:
- Identification of more than 500 high-confidence viral contigs.
- Recovery of complete or near-complete genomes for 36 ancient known phages.
- Discovery of viral taxa consistent across sites and time periods, suggesting long-term stability.

These outcomes significantly surpass the original objectives in terms of the resolution, diversity, and evolutionary depth of ancient phage reconstructions.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The ARCHAIC project delivered several results that extend beyond current virome research standards:

- We systematically evaluated multiple methods for viral detection in ancient metagenomes and demonstrated how different taxonomic classifiers perform under challenging conditions typical of ancient DNA (aDNA), such as short read lengths and damage-induced substitutions. The benchmarking analysis revealed that while genus-level classification is relatively robust across classifiers, species-level annotation remains problematic, especially with comprehensive databases that include bacterial genomes. This insight underscores the need for specialized, virus-focused databases when analyzing ancient samples.

- By applying our optimized pipeline to real ancient datasets from four archaeological sites (Boomerang Shelter, Zape Cave, Arid West Cave, and Hallstatt), we detected over 260 viral species. After rigorous validation through genome breadth coverage and DNA damage profiling, 38 high-confidence viral species were confirmed, all of which were bacteriophages. These findings included both widespread phage taxa and site-specific ones illustrating both ubiquity and local adaptation of ancient phages.

- We also compared assembly-based viral detection using MEGAHIT and metaSPAdes. Although MEGAHIT produced fewer but more complete contigs, both tools contributed to improved viral genome reconstruction when combined with relaxed parameter detection using geNomad. Additionally, our work demonstrated that contig-based analysis could recover viruses missed at the read-level due to sequence degradation, emphasizing the complementary nature of these methods.

Importantly, our approach identified novel viral contigs with no current database match, indicating the presence of ancient viral lineages previously undescribed in modern samples. These findings suggest that ancient samples may harbor unique phage diversity lost in modern times due to changes in lifestyle, environment, or microbiome composition.

Overall, these contributions provide not only a detailed portrait of ancient virome structure and evolution, but also deliver a robust methodological framework for future studies in ancient viral ecology.

Hierarchical workflow for viral detection, and discovery from ancient biological samples